P-selectin is a Ca2+-dependent lectin expressed by activated human platelets and endothelium and is important for mediating rolling adhesion of neutrophils to endothelium in early steps of inflammation. A mucin- like glycoprotein termed P-selectin glycoprotein ligand (PSGL-I) is synthesized by human neutrophils and the promyelocytic cell line HL-60 and appears to be a critical ligand for adhesion of these cells to P- selectin. PSGL- 1 is a disulfide-dimer of 120 kD subunits and contains 2-3 N-linked oligosaccharides and a large number of 0-linked oligosaccharides. In preliminary studies it was found that (a)PSGL-1 contain the sialyl Lewis x (SLex) antigen (NeuAcalpha2-3Galbeta1- 4[Fucalpha1-3]GlcNAcbeta1-) and Lex; (b) the oligosaccharides of PSGL-l are sulfated; (c) 0-linked oligosaccharides of PSGL-1 are larger in size than those CD43 (leukosialin), another mucin-like glycoprotein expressed by neutrophils; and (d) both mono- and polyclonal antibodies have been generated that specifically recognize PSGL-I. Although SLex appears to be an important determinant for neutrophil binding to both P-selectin and E-selectin, preliminary studies demonstrate that purified PSGL-1 binds poorly to E-selectin and that many other neutrophil glycoproteins that do not bind E-selectin also contain the SLex antigen. These results raise questions about the structures of oligosaccharides on PSGL- l and the mechanism of its specific binding to P-selectin. The following 5 specific aims are proposed to provide a greater understanding of the structure and biosynthesis of this important neutrophil glycoprotein. AIM 1. Structurally characterize N- and 0-linked oligosaccharides of PSGL-l from HL-60 cells. AIM 2. Determine the glycans of PSGL- 1 which bind with high affinity to P-selectin and define the important carbohydrate/sulfate residues. AIM 3. Define the biosynthesis of PSGL-1 in HL-60 cells. AIM 4. Study the potential expression of the PSGL-l gene by other cell/tissue types of non-blood origin. AIM 5. Characterize the glycosylation of recombinant forms of PSGL- I from cell lines constructed to have new glycosyltransferases. The long term goals of this work are to understand the mechanism of cell adhesion involving P-selectin and to gain fundamental information about the factors regulating glycoprotein structure/function.